Composition and Structure of the Community of Mycelial Fungi in the Bottom Sediments of the White Sea

Fifty samples of bottom sediments were taken in 2016–2017 in the Kandalaksha Bay of the White Sea from the depth of 1.5–15.0 m, and 1419 colonies of mycelial fungi were obtained. Based on morphological and cultural features, a total of 136 morphotypes were classified, and 81 of these were identified down to the species level. Thirteen species were new to the White Sea. The most common species were Tolypocladium cylindrosporum, Penicillium chrysogenum, Tolypocladium inflatum, Penicillium glabrum, and the anamorph of Pseudogymnoascus pannorum. The dominance of Ascomycota was a common characteristic of the mycobiota due to the anamorphic species, and the class Sordariomycetes was the most diverse and numerous group. Assessment of species richness using a cumulative curve and the calculation of the expected total number of species adjusted using the Chao2 method evidenced that approximately 81% of the species diversity was found within the study. The ordination of samples by the nMDS method with the ANOSIM test showed the high importance of combining the samples into groups based on the year of sampling and type of sediment as well as the year of sampling and type of ecotope. Therefore, the type of sediment associated with the type of coast and the presence of fresh runoff were the most important factors for the formation of mycobiota. Moreover, the communities of mycelial fungi change from year to year in the studied bottom sediments.


INTRODUCTION
Ocean bottom sediments are one of the largest habitats on Earth in terms of coverage [1]. At the same time, until recently, these areas practically did not attract the attention of marine mycologists. It was believed for a long time that only dormant propagules of fungi, brought from land and not participating in the functioning of the ecosystem, could be found here [2]. However, for the last 20 years, the situation has begun to change, and the number of studies of the mycobiota of these habitats is still increasing. Currently, the main attention is focused on studying the diversity of fungi in the bottom sediments of different latitudes [3][4][5][6] and depths [7][8][9][10] as well as on the study of individual features of the isolated fungal strains [11,12]. These studies have demonstrated that marine sediments are a habitat rich in fungi, and their species diversity is often very high. This applies to both coastal [3,[8][9][10][11] and offshore areas [4,7,12]. At present, studies of the fungi diversity in the bottom sediments of the World Ocean and their adaptation to environmental conditions are topical issues of marine mycology [11,12]. Despite the growing scientific interest, the mycobiota of marine sediments has been investigated extremely fragmentarily so far. In addi-tion, in most cases, scientists operate with a small number of samples and do not perform statistical processing of the results. Therefore, the main goal of our study was to identify and to assess the species diversity of the community of filamentous fungi in a small water area and to evaluate the influence of some parameters on its formation using a large number of samples and applying statistical methods.  [3,5,[7][8][9]12]. The samples were started to cultivate directly on the day of collection (or on the next day) on the surface of the wort-agar medium based on natural seawater (0.3% total sugar content, 26‰ water salinity, 0.5 g/L Ceftriaxone antibiotic). Before growing in culture, the material was stored at +6°C. Before inoculation, excess water was poured from the test tube, 1 cm 3 was taken from the sample and evenly distributed with a sterilized spatula over five plates with the medium. They were incubated at +6°C for 6 weeks, after which the colonies were isolated into a pure culture. The isolated strains were identified down to the species level whenever possible. If this was impossible, then these colonies were attributed to the separate morphotypes (some morphotypes were represented by several colonies). The names and taxonomic position of the fungi were specified using the database www.indexfungorum.org/Names/fungic.asp.

Sampling
Statistical processing. The total number of colonies and the number of colonies of each species and morphotype were counted on each plate. These data were then used to calculate the total number of colonies and the number of colonies of each species and morphotype in individual samples. Shannon and Pielu indices were calculated to determine the actual species diversity and evenness. Species richness was assessed by the cumulative curve of species number accumulation with increasing number of samples. Since the obtained curve did not reach a plateau (an increase in the number of samples led to an increase in the number of species), we calculated the expected total number of species using Chao2 bias correction for rare species [13]. The degree of similarity of the fungal communities from individual samples was analyzed using the Bray-Curtis similarity index and the Sørensen similarity index. The similarity matrices were then used for sample ordination by the method of nonmetric multidimensional scaling (nMDS) to identify general trends in the distribution of communities. The results of ordination were checked and supplemented by the method of one-way analysis of similarity (ANOSIM), which allowed assessing the reliability of combining samples into certain groups [13,14]. The grouping was considered nonrandom if the threshold level of significance did not exceed 5%. The samples were grouped as following: (1) year of selection (2016/2017); (2) type of sediment (lightly silted/heavily silted); (3) depth (less than 5 m/5-10 m/more than 10 m); (4) type of ecotope (open shore without fresh runoff/open shore with fresh runoff/semienclosed bight with fresh runoff/enclosed bay with fresh runoff); (5) year + type of sediment; (6) year + type of ecotope. The contribution of particular species to intragroup similarity and the identification of species responsible for grouping (responsible for combining samples into groups) were assessed using the SIMPER procedure. The packages MS Excel 2007 (Microsoft™) and PRIMER v6 (Primer™, 2001) were used for all calculations.

RESULTS AND DISCUSSION
General information about the number and diversity of mycobiota. In total, 1419 colonies of filamentous fungi grew in all dishes (Table 1). Seven to seventyfour, in most cases, 20-50, colonies were isolated from 1-cm 3 samples. This number is relatively small. It is slightly lower than the number of fungi in silty sediments at the depths of 54-108 m in the Velikaya Salma Strait [5] and by 1-2 orders of magnitude lower than the number obtained by A. Khusnullina et al. [10] from pebble-silty-sandy sediments at the depths of 0-30 m in the same research area. Presumably, the latter may be explained by different methodological approaches. The relatively low abundance of fungal propagules obtained in the present study may also be associated with the type of sediment: most of the studied samples were lightly silted sandy sediment, as a rule, the abundance of fungi in such type of bottom sediments is somewhat lower than in heavily silty sediments [15].
The diversity of the mycobiota of the studied sediments is high, but the abundance is relatively low. On the basis of morphological and cultural features, 136 morphotypes were isolated. We identified 81 to the species level and 14 to the genus level, while 43 were not identified because they were sterile. Six to twentyfour morphotypes were isolated from one sample, in most cases, 10-20. A number of species that were not previously known for the White Sea were identified [10,15,16]. Namely, these are twelve species belonging to 11 genera of anamorphic Ascomycota: Acremonium alternatum Link; Aspergillus ochraceus G. Wilh.;  [17]. All other species are also known from the soils and freshwater habitats, most of them, in the vicinity of WSBS [16].
Tolypocladium cylindrosporum W. Gams (227 colonies/36 samples), Penicillium chrysogenum Thom (123/40), T. inflatum W. Gams (87/27), Penicillium glabrum (Wehmer) Westling (86/25), and Pseudogymnoascus pannorum (Link) Minnis & DL Lindne (54/28; four isolates with fruiting bodies, the rest with only conidia) were the most common species found in at least 50% of the samples and they formed a relatively large number of colonies. Another ten species were found in at least ten samples. The other morphotypes, constituting the most part (121), were found in less than ten samples; many of them were represented by single colonies in separate samples. Penicillium was the most diverse and widely represented at the genus level (17 species and one unidentified morphotype, 392 colonies in 48 samples) followed by Acremonium (eight species and four unidentified morphotypes, 108 colonies in 31 samples) and Trichoderma (six species and three unidentified morphotypes, 45 colonies in 19 samples). The genus Tolypocladium, which is characterized by less diversity, had a high abundance and frequency of occurrence (329 colonies/45 samples). One hundred and thirty-three sterile colonies were isolated from 43 samples. According to morphological and cultural features, they were divided into 43 morphotypes, of which 26 were light-colored and 17 were melanized. In general, light-colored forms also predominated in the mycobiota.
Ascomycota absolutely predominated in the taxonomic structure of mycobiota (Table 1): their share in the total number of isolated fungal strains was at least 89.2%. In turn, Eurotiomycetes and Sordariomycetes were the most abundant classes of this phylum. Sordariomycetes was the most diverse in terms of all the taxonomic levels, from morphotypes to orders. The total number of isolated Zygomycota was extremely small, when only 19 colonies representing six species were grown from 11 samples.
In general, this structure of the mycobiota was not a big surprise. It has long been known that Ascomycota predominate in marine ecotopes and their anamorphic forms predominate in culture [2,17]. The predominance of Ascomycota in marine ecotopes has been shown using not only direct and cultural methods but also molecular approach [7,11,18]. Previous studies of the mycobiota of the bottom sediments of the White Sea, as well as the bottom sediments of lakes separating from the White Sea, also showed the predominance of anamorphic Ascomycota, a high proportion of representatives of the genera Penicillium and Tolypocladium, and the predominance of light-colored forms [5,10,19]. Similar features are known for the sediments of other cold-water seas [6,9,12]. A high abundance of Ascomycota (particularly, class Sordariomycetes) was a noticeable distinctive feature of the mycobiota of the samples studied within the present work. In previous studies performed in the vicinity of WSBS [5,10], this group was presented as less diverse compared to Eurotiomycetes. Thus, the mycobiota described within the present study has many features characteristic of the previously studied fungal communities of bottom sediments of various cold-water seas. High abundance and high diversity of representatives of the class Sordariomycetes was its main distinguishing feature.     [3,9]. The Pielu index was from 0.59 to 0.96 in particular samples, averaging 0.88, which was also relatively high.
The cumulative curve of species accumulation does not reach a plateau (Fig. 1); this indicates that the mycobiota diversity is not described in full in our study. In addition, the curve has a smooth increase; therefore, more samples must be used to identify species richness. For example, the processing of 20 samples revealed approximately 50% of the total species richness, while that of 30 samples revealed approximately 70%. The calculation with the Chao2 correction evidences that the expected total number of species in the study area must be 167. Therefore, in our work, the revealed diversity is approximately 81% of the theoretically possible. It should be noted that no studies in the field of marine mycology using such statistical assessment of species diversity are known to date. However, given that the number of samples is 10-20, rarely approximately 30, in most studies on the diversity of benthic mycobiota, it can be assumed that this may be the reason for the overall low level of knowledge of the mycobiota of marine sediments along with a relatively small number of studies in general.
The similarity indices for individual samples were relatively low; the Sørensen index was slightly higher (from 7.1 to 64.7%, 32.4% on average), the Bray-Curtis index was slightly lower (from 5.3 to 57.8%, 28.9% on average). However, further nMDS analysis demonstrated a significant grouping of samples according to a number of parameters in both cases. The level of significance of the grouping reliability for the Sørensen index according to ANOSIM was 0.2 for the year of sampling, 0.6, for type of sediment, 37.6, for depth, 1.7, for type of ecotope, and 0.1 for both type of sediment + year of sampling and type of ecotope + year of sampling. For the Bray-Curtis index, the pattern was fundamentally the same. Therefore, the combination of samples is unreliable only in terms of the depth of sampling, while the best solution is the combining the year of sampling with the sediment type and the ecotope type. The type of ecotope in most cases is associated with the type of sediment: siltation occurs either in more enclosed areas of the coast or in areas with fresh runoff; i.e., the type of sediment associated with the type of coast and the presence of fresh water is the most important factor for the formation of mycobiota. In addition, significant changes may occur in the mycobiota of bottom sediments from year to year, which is shown by the example of 2-year observations in the present study. Obviously, these differences may be associated with a variety of weather factors that were not possible to isolate and to study separately within the framework of this study. A total of 17 species may be responsible for the grouping ( Table 2), but their distribution and proportions depend on a particular group but depend weakly on the particular value of the similarity index. For example, as the sediment siltation increases, the representatives of the genus Tolypocladium disappear from the community of dominant species, but the diversity of Penicillium increases. In addition, the diversity of Cephalosporium-like fungi and the overall diversity of the group of species responsible for grouping are increasing. The results are also influenced by the year of the study. For example, Pseudogymnoascus pannorum was one of the species responsible for grouping on the open shores without fresh runoff in 2016, but it totally disappeared in 2017, although these communities were in fact similar. In silted areas, the difference over the years is even more pronounced. Therefore, we attempted to assess the species diversity and structure of the mycobiota of bottom marine  sediments using the methods of statistical analysis. This approach is definitely useful since it allows a clearer understanding of the results of the fungi growing in culture. Using this approach, it is possible to confidently determine the influence of the type of sediment and type of ecotope on the composition and structure of the marine benthic mycobiota; it also allows one to analyze the general differences in the composition and structure of mycobiota in different years of research as well as to assess the number of samples required for the most complete identification of species diversity.

ACKNOWLEDGMENTS
The authors express their gratitude to the scuba-diving team of WSBS for help in organizing the collection of research materials and to M.V. Chikina (Shirshov Institute of Oceanology, Russian Academy of Sciences) for a discussion of the study results.

FUNDING
The study was carried out with partial financial support of the Russian Foundation for Basic Research (grant no. 20-04-00882a, processing and analysis of primary data on species composition) and the grant of Moscow State University for supporting the leading scientific schools of Moscow State University "'Noah's Ark' Living Systems Depository, National Depositary Bank of Living Systems" in the framework of the Development Program of Moscow State University (statistical processing).

COMPLIANCE WITH ETHICAL STANDARDS
Statement on the welfare of animals. This article does not contain any studies involving animals or human participants performed by any of the authors.